Special Seminar: Paul Gooley

The ectodomain of the relaxin GPCR, RXFP1, comprises an N-terminal LDLa module, essential for activation, tethered to a leucine-rich repeat (LRR) domain by a 32-residue linker. Activation is thought to proceed by relaxin binding with strong affinity to the LRR domain and then, through an unknown process, enable the LDLa module, proposed to be a tethered agonist, to bind and activate the transmembrane domain. While the linker has been dismissed as simply a disordered region to tether the LDLa module to the receptor, we have found mutations within a conserved region significantly weakens relaxin affinity, suggesting an additional binding site. Using NMR spectroscopy and titrations of 15N-labelled LDLa-linker with relaxin or a paramagnetic (Mn2+) labeled relaxin, we have elucidated a discrete relaxin-binding site (residues 46-63) on the linker. Based on these, and additional NMR experiments, we hypothesize that LDLa-LRR linker and relaxin binding is a two-step mechanism in which partially ordered conformations of the linker form a complex with relaxin and then rapidly rearrange to form a stable helical structure, which then serves as the true agonist for receptor activation.